LC-MS-MS aboard ship: tandem mass spectrometry in the search for phycotoxins and novel toxigenic plankton from the North Sea

Phycotoxins produced by various species of toxigenic microalgae occurring in the plankton are a global threat to the security of seafood resources and the health of humans and coastal marine ecosystems. This has necessitated the development and application of advanced methods in liquid chromatography coupled to mass spectrometry (LC-MS) for monitoring of these compounds, particularly in plankton and shellfish. Most such chemical analyses are conducted in land-based laboratories on stored samples, and thus much information on the near real-time biogeographical distribution and dynamics of phycotoxins in the plankton is unavailable. To resolve this problem, we conducted ship-board analysis of a broad spectrum of phycotoxins collected directly from the water column on an oceanographic cruise along the North Sea coast of Scotland, Norway, and Denmark. We equipped the ship with a triple-quadrupole linear ion-trap hybrid LC-MS-MS system for detection and quantitative analysis of toxins, such as domoic acid, gymnodimine, spirolides, dinophysistoxins, okadaic acid, pectenotoxins, yessotoxins, and azaspiracids (AZAs). We focused particular attention on the detection of AZAs, a group of potent nitrogenous polyether toxins, because the culprit species associated with the occurrence of these toxins in shellfish has been controversial. Marine toxins were analyzed directly from size-fractionated plankton net tows (20 μm mesh size) and Niskin bottle samples from discrete depths, after rapid methanolic extraction but without any further clean-up. Almost all expected phycotoxins were detected in North Sea plankton samples, with domoic acid and 20-methylspirolide G being most abundant. Although AZA was the least abundant of these toxins, the high sensitivity of the LC-MS-MS enabled detailed quantification, indicating that the highest amounts of AZA-1 were present in the southern Skagerrak in the 3–20 μm size-fraction. The direct on-board toxin measurements enabled isolation of plankton from stations with high AZA-1 levels and from the most suspicious size-fraction, i.e. most likely to contain the AZA-producer. A large number (>100) of crude cultures were established by serial dilution and later screened for the presence of AZAs after several weeks growth. From one crude culture containing AZA, a small dinoflagellate was subsequently isolated and brought into pure culture. We have thus proved that even sophisticated mass spectrometers can be operated in ship laboratories without any limitation caused by vibrations of the ship’s engine or by wave movement during heavy seas at wind forces up to nine Beaufort. On-board LC–MS–MS is a valuable method for near real-time analysis of phycotoxins in plankton for studies on bloom dynamics and the fate of toxins in the food web, and for characterization and isolation of putatively toxigenic organisms.     

Development and validation of a sensitive enzyme immunoassay for surveillance of Cry1Ab toxin in bovine blood plasma of cows fed Bt-maize (MON810)

The increasing global adoption of genetically modified (GM) plant derivatives in animal feed has provoked a strong demand for an appropriate detection method to evaluate the existence of transgenic protein in animal tissues and animal by-products derived from GM plant fed animals. A highly specific and sensitive sandwich enzyme immunoassay for the surveillance of transgenic Cry1Ab protein from Bt-maize in the blood plasma of cows fed on Bt-maize was developed and validated according to the criteria of EU-Decision 2002/657/EC. The sandwich assay is based on immuno-affinity purified polyclonal antibody raised against Cry1Ab protein in rabbits. Native and biotinylated forms of this antibody served as capture antibody and detection antibody for the ELISA, respectively. Streptavidin-horseradish peroxidase conjugate and TMB substrate provided the means for enzymatic colour development.The immunoassay allowed Cry1Ab protein determination in bovine blood plasma in an analytical range of 0.4-100 ng mL⁻¹ with a decision limit (CCα) of 1.5 ng mL⁻¹ and detection capability (CCβ) of 2.3 ng mL⁻¹. Recoveries ranged from 89 to 106% (mean value of 98%) in spiked plasma.In total, 20 plasma samples from cows (n = 7) fed non-transgenic maize and 24 samples from cows (n = 8) fed transgenic maize (collected before and, after 1 and 2 months of feeding) were investigated for the presence of the Cry1Ab protein. There was no difference amongst both groups (all the samples were below 1.5 ng mL⁻¹; CCα). No plasma sample was positive for the presence of the Cry1Ab protein at CCα and CCβ of the assay.     

Solid-phase extraction-fluorimetric high performance liquid chromatographic determination of domoic acid in natural seawater mediated by an amorphous titania sorbent

The feasibility of using sol-gel amorphous titania (TiO₂) as a solid-phase sorbent for the pre-concentration of domoic acid (DA), a potent amnesic shellfish poisoning (ASP) toxin, directly from seawater was explored. The sol-gel titania material is able to adsorb DA from seawater, via the formation of ester-linkage between the carboxylic moieties of DA and the Ti-OH groups on the sorbent surface, at low pH and desorb it at high pH. The chemisorption process is not significantly interfered by the seawater matrix. The optimum pH values for the adsorption and desorption of DA were found to be pH 4 and 11, respectively. The optimal sorbent loading for the batch-type solid-phase extraction of DA was 0.67 mg-TiO₂ ng-DA⁻¹ and adsorption equilibrium was achieved in 2 h at room temperature. The desorbed DA in 500 μL of 0.1 M alkaline borate buffer can be directly derviatized by 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) in aqueous media for fluorimetric HPLC quantification. Analyte recovery, repeatability and detection limit of this titania SPE-fluorimetric HPLC determination are 89%, 6.2% and 120 pg-DA mL⁻¹ (n = 7, P < 0.05), respectively, for a sample volume of 30 mL. This titania SPE technique should also be applicable to the pre-concentration of other polar carboxylate- and phosphonate-containing biomolecules and pharmaceuticals in complex and interfering environmental sample matrices.     

新型蛋白结合类毒素血液灌流吸附剂的制备及吸附性能

蛋白结合类毒素(PBUT)在尿毒症并发症的发生发展中起着重要作用, 现有血液净化模式对其清除效果较差, 开发用于高效清除尿毒症患者体内PBUT的血液灌流吸附材料已成为迫切的临床需求. 本文首先采用悬浮聚合法制备了咪唑基改性低交联聚苯乙烯微球P(St-DVB-VMZ); 然后通过小分子外交联剂的一步法傅克烷基化后交联反应, 制备出血液灌流用含咪唑基超高交联聚苯乙烯多孔树脂吸附剂HCP(St-DVB-VMZ). 利用傅里叶变换红外光谱(FTIR)、 X射线光电子能谱(XPS)、 扫描电子显微镜(SEM)及N₂吸附-脱附分析等表征了吸附树脂的化学结构和微观孔结构. 结果表明, HCP(St-DVB-VMZ)具有丰富的孔结构, 比表面积达到709 m²/g. 尿毒症毒素吸附实验结果表明, HCP(St-DVB-VMZ)对蛋白结合类毒素[对硫酸吲哚酚(IS)、 对甲酚硫酸盐(PCS)和吲哚乙酸(IAA)]和中大分子毒素[甲状旁腺激素(PTH)、 β₂-微球蛋白(β₂M)及白细胞介素6(IL-6)]均具有优异的吸附性能并展示出较好的血液相容性, 有望实现全血灌流临床应用.     

Identification and quantification of domoic acid by UHPLC/QTOF tandem mass spectrometry,with simultaneous identification of non-target photodegradation products

Amnesic shellfish poisoning is a potentially lethal human toxic syndrome which is caused by domoic acid (DA), a neurotoxin produced by marine phytoplankton, principally from Pseudonitzschia genus. In this report, a method to identify and quantify the DA toxin, with simultaneous identification of its photodegradation products, has been developed. It uses an ultra high performance liquid chromatography coupled to a quadrupole-time-of-flight tandem mass spectrometer (UHPLC–QTOF) after solid-phase extraction (SPE). An unambiguous identification of DA was carried out by considering both the retention time of DA in UHPLC and the exact mass of protonated DA molecule ([M + H]⁺ = 312.1447 m/z) and of the most intense fragment ion (m/z 266.1391). The quantification was conducted using protonated DA molecule with protonated Glafenin as internal standard, obtaining a limits of detection of 0.75 µg L^?1. Large screening with UHPLC–QTOF could also give structural information about degradation products of DA present in samples after UV-irradiation. This method was applied for the determination of DA in complex liquid samples after SPE and is applicable for environmental monitoring of this toxic substance in the aquatic environment.     

超高效液相色谱-串联质谱法测定水华样品中微囊藻毒素及其基质效应的消除

超高效液相色谱-串联质谱法(UPLC-MS/MS)由于其快速、简便的特点,目前广泛应用于水中微囊藻毒素的测定。由于蓝藻水华样品基质比较复杂,UPLC-MS/MS在复杂基质的分析过程中容易发生基质效应问题,严重影响其定量分析结果的准确性。通过稀释样品、优化梯度洗脱程序、减少进样量等方法有效地降低或消除了基质干扰及基质效应问题。7种目标物在各自线性范围内线性关系良好,相关系数(r2)≥0.99;检出限和定量限分别为0.064~0.103μg/L和0.213~0.343μg/L。通过稀释样品、减少进样量等措施,成功测定了水华爆发时实际样品中的微囊藻毒素,并有效降低了基质效应的干扰。该法为微囊藻毒素的准确测定提供一定的参考,对保障饮用水源地的安全具有十分重要的意义。     

Characterization of (<Superscript>13</Superscript>C<Subscript>24</Subscript>) T-2 toxin and its use as an internal standard for the quantification of T-2 toxin in cereals with HPLC–MS/MS

In this paper, the structure and the identity of fully ¹³C-substituted T-2 toxin were confirmed using high-resolution mass spectrometry, ¹H-NMR, ¹³C-NMR, tandem mass spectrometry and HPLC–DAD. The purity of this compound was estimated to be at least 98.8% according to UV data. The isotopic distribution of (¹³C₂₄) T-2 toxin indicated a total isotopic enrichment of 98.2 ± 1.0 atom% ¹³C, and the application of different MS measurement modes revealed the MS/MS fragmentation pattern of T-2 toxin. Furthermore, a stable isotope dilution mass spectrometry method for the quantification of T-2 toxin was developed using (¹³C₂₄) T-2 toxin as internal standard. The method was evaluated with and without conventional clean-up and validated for maize and oats. Both cereals showed strong matrix enhancement effects, which could be compensated for through the application of the isotope-substituted internal standard.     

Rapid and sensitive detection of cholera toxin using gold nanoparticle-based simple colorimetric and dynamic light scattering assay

Herein, a rapid and simple gold nanoparticle based colorimetric and dynamic light scattering (DLS) assay for the sensitive detection of cholera toxin has been developed. The developed assay is based on the distance dependent properties of gold nanoparticles which cause aggregation of antibody-conjugated gold nanoparticles in the presence of cholera toxin resulting discernible color change. This aggregation induced color change caused a red shift in the plasmon band of nanoparticles which was measured by UV–Vis spectroscopy. In addition, we employed DLS assay to monitor the extent of aggregation in the presence of different concentration of cholera toxin. Our assay can visually detect as low as 10 nM of cholera toxin which is lower than the previously reported colorimetric methods. The reported assay is very fast and showed an excellent specificity against other diarrhetic toxins. Moreover, we have demonstrated the feasibility of our method for cholera toxin detection in local lake water.     

Exploring Multi-Subsite Binding Pockets in Proteins: DEEP-STD NMR Fingerprinting and Molecular Dynamics Unveil a Cryptic Subsite at the GM1 Binding Pocket of Cholera Toxin B

Ligand-based NMR techniques to study protein–ligand interactions are potent tools in drug design. Saturation transfer difference (STD) NMR spectroscopy stands out as one of the most versatile techniques, allowing screening of fragments libraries and providing structural information on binding modes. Recently, it has been shown that a multi-frequency STD NMR approach, differential epitope mapping (DEEP)-STD NMR, can provide additional information on the orientation of small ligands within the binding pocket. Here, the approach is extended to a so-called DEEP-STD NMR fingerprinting technique to explore the binding subsites of cholera toxin subunit B (CTB). To that aim, the synthesis of a set of new ligands is presented, which have been subject to a thorough study of their interactions with CTB by weak affinity chromatography (WAC) and NMR spectroscopy. Remarkably, the combination of DEEP-STD NMR fingerprinting and Hamiltonian replica exchange molecular dynamics has proved to be an excellent approach to explore the geometry, flexibility, and ligand occupancy of multi-subsite binding pockets. In the particular case of CTB, it allowed the existence of a hitherto unknown binding subsite adjacent to the GM1 binding pocket to be revealed, paving the way to the design of novel leads for inhibition of this relevant toxin.